EP0848753B1 - Nukleinsäuremolekül, das für einen tgf-rezeptor mit veränderter wachstumshemmung kodiert und seine verwendung - Google Patents

Nukleinsäuremolekül, das für einen tgf-rezeptor mit veränderter wachstumshemmung kodiert und seine verwendung Download PDF

Info

Publication number
EP0848753B1
EP0848753B1 EP96929418A EP96929418A EP0848753B1 EP 0848753 B1 EP0848753 B1 EP 0848753B1 EP 96929418 A EP96929418 A EP 96929418A EP 96929418 A EP96929418 A EP 96929418A EP 0848753 B1 EP0848753 B1 EP 0848753B1
Authority
EP
European Patent Office
Prior art keywords
tβr
type
tgf
nucleic acid
receptor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP96929418A
Other languages
English (en)
French (fr)
Other versions
EP0848753A2 (de
Inventor
Masae The Cancer Institute SAITOH
Kohei The Cancer Institute MIYAZONO
Hidenori The Cancer Institute ICHIJO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ludwig Institute for Cancer Research Ltd
Ludwig Institute for Cancer Research London
Ludwig Institute for Cancer Research New York
Original Assignee
Ludwig Institute for Cancer Research Ltd
Ludwig Institute for Cancer Research London
Ludwig Institute for Cancer Research New York
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ludwig Institute for Cancer Research Ltd, Ludwig Institute for Cancer Research London, Ludwig Institute for Cancer Research New York filed Critical Ludwig Institute for Cancer Research Ltd
Publication of EP0848753A2 publication Critical patent/EP0848753A2/de
Application granted granted Critical
Publication of EP0848753B1 publication Critical patent/EP0848753B1/de
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a nucleic acid molecule encoding a type I receptor of the TGF- ⁇ superfamily having modified growth inhibition, and its use.
  • TGF- ⁇ transforming growth factor- ⁇
  • BMP bone morphogenetic protein
  • Mullerian-inhibiting substance glial cell line-derived neurotrophic factor.
  • TGF- ⁇ is a prototype in this superfamily of structurally related molecules, and regulates cell proliferation, extracellular matrix formation, migration, adhesion and many other cellular functions important for development and homeostasis.
  • TGF- ⁇ superfamily exert their biological actions through heteromeric complexes of two types (type I and type II) of transmembrane receptors with a serine/threonine kinase domain in their cytoplasmic region.
  • type I receptors six different type I receptors have been identified in mammals (ten Dijke et al (1994) Prog. Growth Factor Res. 5 :55-72), including one TGF- ⁇ type I receptor (T ⁇ R-I), two activin type I receptors (ActR-I and ActR-IB), two BMP type I receptors (BMPR-IA and BMPR-IB) and one additional type I receptor called ALK-1.
  • type I receptors have similar sizes (502-532 amino acid residues) and 60-90% amino acid sequence identities to each other in their kinase domains.
  • type I receptors contain a conserved sequence known as the GS domain (also called type I box) in their cytoplasmic juxtamembrane region (Attisano et al (1994) Biochem. Biophys. Acta 1222:71-80).
  • Type I receptors are more similar to each other than they are to the known type II receptors including TGF- ⁇ type II receptor (T ⁇ R-II) and two activin type II receptors (ActR-II and ActR-IIB), and thus form a subgroup of mammalian type I receptors in the family of receptor serine/threonine kinases.
  • T ⁇ R-II TGF- ⁇ type II receptor
  • ActR-II and ActR-IB activin type II receptors
  • T ⁇ R-II is a constitutively active kinase and capable of binding TGF- ⁇ in the absence of T ⁇ R-I, whereas T ⁇ R-I requires T ⁇ R-II for the ligand-binding.
  • the T ⁇ R-I kinase appears to be activated by formation of a hetero-oligomeric complex composed of TGF- ⁇ , T ⁇ R-II and T ⁇ R-I.
  • T ⁇ R-I acts as a downstream signalling molecule of T ⁇ R-II (Wieser et al (1995) supra).
  • the GS domain for initiating intracellular signals, little is known about how the signals are propagated after phosphorylation of the GS domain.
  • the phosphorylated serine and/or threonine residues in the GS domain may act as the binding sites for the intracellular substrate to be activated by the T ⁇ R-I kinase. This hypothesis is attractive to explain the signalling mechanism for certain common effects induced by the members of TGF- ⁇ superfamily since the GS domain of the known type I receptors is highly conserved.
  • amino acid sequences of the GS domain of the type I receptors might be too similar to each other to confer specificities to the signals which mediate a wide variety of responses induced by the TGF- ⁇ superfamily.
  • a T ⁇ R-I chimeric receptor substituting ActR-I GS domain for T ⁇ R-I GS domain still has the TGF- ⁇ -induced antiproliferative signal which is not mediated through intact ActR-I (Wieser et al (1995) supra) .
  • certain region(s) other than the GS domain in the type I receptors may also be important for diverse signalling activities of the TGF- ⁇ superfamily.
  • TGF- ⁇ growth inhibition and matrix formation by TGF- ⁇ are potent, and physiologically important. However, these effects are not always desirable during the process of human diseases. For example, cancer cells grow autonomously, and therefore the growth inhibitory effect of TGF- ⁇ is important. Matrix formation induced by TGF- ⁇ is undesirable, because it induces fibrosis of the tissues, which is found in certain cancer tissues, i.e. gastric cancer and hepatoma. Thus, matrix formation by TGF- ⁇ is preferably inhibited, in the treatment of cancer, while keeping the growth inhibitory activity of TGF- ⁇ intact.
  • novel TGF receptors are mutated such that they substantially retain their matrix formation effect, and exhibit reduced (e.g. to a level no more than 50%, preferably no more than 25%) growth inhibitory effect.
  • the invention relates also to any fragment of such materials which retain these characteristics, and corresponding nucleotides.
  • the new T ⁇ R-I mutants are useful to identify intracellular substrates which transduce the growth inhibitory signal but not the matrix formation signal.
  • the mutants are also useful for screening drugs that can be used for these purposes.
  • the mutants are also of utility in making transgenic mammals such as mice, the consequence of which may be important to understand the growth inhibitory activity and matrix formation activity in vivo, by comparison with wild-type T ⁇ R-I and kinase-deficient mutants.
  • T ⁇ R-I cytoplasmic juxtamembrane region located between the transmembrane domain and the GS domain of T ⁇ R-I by mutational analyses using mutant mink lung epithelial cells which lack endogenous T ⁇ R-I.
  • wild-type T ⁇ R-I restored the TGF- ⁇ signals for growth inhibition and induction of plasminogen activator inhibitor (PAI)-1 and fibronectin.
  • PAI plasminogen activator inhibitor
  • a deletion mutant, T ⁇ R-I/JD1( ⁇ 150-181) which lacks the juxtamembrane region preceding the GS domain, bound TGF- ⁇ in concert with T ⁇ R-II and transduced a signal leading to induction of PAI-1 and fibronectin but not growth inhibition.
  • Recombinant receptors with mutations that change serine 172 to alanine (S172A) or threonine 176 to valine (T176V) were similar to wild-type T ⁇ R-I in their abilities to bind TGF- ⁇ , formed complexes with T ⁇ R-II, and transduced a signal for PAI1 and fibronectin. Similarly to T ⁇ BR-I/JD1( ⁇ 150-181), however, these missense mutant receptors were impaired in their effect to mediate a growth inhibitory signal.
  • oligonucleotides see also the Sequence Listing; SEQ ID Nos. 1-12 were used to generate expression constructs.
  • the sequences of the oligonucleotide primers are presented below in the 5' to 3' direction. Numbering is based on the nucleotide sequence of T ⁇ R-I (Franzen et al (1993) Cell 75 :681-692). Restriction enzyme sites incorporated into the primers are underlined. The junction of the deletion primer RISde15 is indicated by a -.
  • cDNA Constructions Stable expression vectors of wild-type T ⁇ R-I and its mutant derivatives were prepared by subcloning PCR-generated cDNA fragments into pMEP4 vector, a Zn 2+ -inducible mammalian expression vector (Wrana et al (1992) Cell 71 :1003-1014).
  • pMEP4 vector a Zn 2+ -inducible mammalian expression vector
  • primer RISO-hind and primer RIAS-not were used to amplify the coding region of T ⁇ R-I cDNA. Reaction conditions were 1 min at 94°C, 1 min at 48°C, and 2 min at 72°C for 30 cycles.
  • the PCR products were digested with HindIII and Not I, and subcloned into the pMEP4 vector.
  • the primers RIS0-hind and RIASdel1 were used to amplify the 5' part of T ⁇ R-I cDNA fragment, and the primers RISdel5 and RIAS-not were used for the 3' fragment.
  • the two primary PCR products were gel-purified, mixed and subjected to reamplification with primers RISO-hind and RIAS-not.
  • the secondary PCR products were digested with HindI II and Not I, and subcloned into the pMEP4 vector.
  • primer RISO-hind and the mutant antisense primer were used to amplify the 5' fragments
  • the mutant sense primer S-1, S-2 and S-3, respectively
  • primer RIAS-not were used to amplify the 3' fragments.
  • PCR products were mixed in respective combinations, and reamplified with primers RISO-hind and RIAS-not.
  • PCR was performed using T ⁇ R-I/JMI as a template for the 5' fragment with primers RIS0-hind and AS-2, and T ⁇ R-I/JM3 as a template for the 3' fragment with primers S-2 and RIAS-not.
  • the two PCR fragments were mixed and reamplified with primers RIS0-hind and RIAS-not.
  • the Sma I -Xba I fragments of the mutant PCR products were swapped for the corresponding region of wild-type T ⁇ R-I plasmid.
  • Expression vectors for bacterial expression of wild-type T ⁇ R-I glutathione S-transferase (GST) fusion proteins (GST-WT), its deletion mutant GST-JD1( ⁇ 150-181) and missense mutants GST-JM1(S165A), GSTJM2 (S172A), GST-JM3(T176V) and GST-JM1 23(S165A/S172A/T176V), were obtained by insertion of PCR-generated fragments of the corresponding cytoplasmic regions of T ⁇ R-I into pGEX-4T-1 (Pharmacia) using their stable expression plasmids as templates with RIS1-sma or RISdel2-sma as sense primers and RIAS-not as an antisense primer.
  • GST glutathione S-transferase
  • PCR conditions were 1 min at 94°C, 1 min at 54°C, and 1 min at 72°C for 25 cycles.
  • the resulting PCR products for the GST fusion protein constructs were digested with Sma I and Not I, and ligated in-frame into pGEX-4T-1.
  • R4-2 cells were transfected by the calcium phosphate precipitation method using Eukaryotic transfection kit (Promega). Selection of transfected cells was performed in the presence of 120 U/ml of hygromycin B (Wako Chemicals). Resistant cell colonies were examined for the expression of T ⁇ R-I and its mutants by the receptor affinity-labelling assays using 125 ITGF- ⁇ 1 after induction of the recombinant proteins by ZnCl 2 . More than two independent clones for each of the transfectants were subjected to the following experiments.
  • the ligand-receptor complexes were cross-linked with 0.27 mM DSS (Pierce Chemical Co.).
  • Cells were washed once with 10 mM Tris-HCl (pH 7.4) containing 1 mM EDTA and 10% glycerol, and solubilized by incubation in TNE buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40) containing 1.5% of aprotinin for 20 min at 4°C.
  • PAI-1 Assay- PAI-1 assays were performed as previously described with minor modifications (Carcamo et al (1994) Mol. Cell. Biol. 14 :3810-3821) . Briefly, subconfluent cells in 6-well plates were incubated for 5 h with DMEM containing 0.2% FBS and 100 ⁇ M ZnCl 2 .
  • the cells were then removed by washing once in PBS, four times in 10 mM Tris-HCl, pH 8.0, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, two times in 2 mM Tris-HCl, pH 8.0, and once in PBS.
  • Proteins were extracted from plastics by SDS-sample buffer containing 10 mM DTT and were analyzed by SDS-10% polyacrylamide gel electrophoresis and Bio-Imaging Analyzer. Fibronectin Assay - Measurement of fibronectin was performed as described with minor modifications (Wrana et al (1992) supra).
  • the beads were washed once in Tris-buffered saline (50 mM Tris-HCl, pH 7.4, 150 mM NaCl), once in 50 mM Tris-HCl (pH 7.4), 0.5 M NaCl, and once in Tris-buffered saline.
  • the fibronectin was eluted by boiling in SDS-sample buffer in the presence of 10 mM DTT.
  • the samples were analyzed by SDS-7% polyacrylamide gel electrophoresis and Bio-Imaging Analyzer.
  • Protein Kinase Assay 25 ⁇ l glutathione-Sepharose beads which attached GST fusion proteins were washed once with kinase buffer (20 mM Hepes, pH 7.4, 100 mM NaCl, 10 mM MnCl 2 , 0.5 mM DTT, 0.05% Triton X100). 25 ⁇ l kinase buffer containing 1 ⁇ Ci of [ ⁇ - 32 P]ATP (Amersham) was added. The beads were incubated for 15 min at 4°C. Proteins were resolved on SDS-10% polyacrylamide gel under reducing conditions and analyzed by Bio-Imaging Analyzer.
  • T ⁇ R-I/JD1( ⁇ 150-181) 32 amino acids of T ⁇ R-I in the juxtamembrane region were deleted, yielding T ⁇ R-I/JD1( ⁇ 150-181).
  • the expression of the exogenous receptors and their complex formation with the endogenous T ⁇ R-II were tested by affinity cross-linking of the cells using 125 ITGF- ⁇ 1 followed by immunoprecipitating the ligand-receptor complexes with anti-T ⁇ R-II antiserum.
  • T ⁇ R-I/JD1( ⁇ 150-181) was able to bind TGF- ⁇ in a Zn 2+ -inducible manner and form a physiological complex with
  • T ⁇ R-I/JD1( ⁇ 150-181) The signalling activities of T ⁇ R-I/JD1( ⁇ 150-181) were determined by testing its ability to rescue biological responses to TGF- ⁇ in R4-2 cells.
  • the induction of PAI-1 and fibronectin were examined since these responses in the parent Mv1Lu cells are well-characterized and are representative of the various matrix proteins induced by TGF- ⁇ .
  • synthesis of PAI-1 was increased by the treatment with TGF- ⁇ , but not in R4-2 cells transfected with the vector alone.
  • R4-2 cells were transfected with the wild-type T ⁇ R-I or T ⁇ R-I/JD1( ⁇ 150-181), the cells produced PAI-1 upon treatment with TGF- ⁇ in the presence of ZnCl 2 .
  • fibronectin production by TGF- ⁇ was restored in R4-2 cells transfected with the wild-type T ⁇ R-I and less potently in the cells transfected with T ⁇ R-I/JD1( ⁇ 150-181).
  • PAI-1 and fibronectin production were not stimulated in the absence of ZnCl 2 , indicating that the signals for the induction of PAI-1 and fibronectin were rescued by the exogenous receptors.
  • T ⁇ R-I/JD1( ⁇ 150-181) is able to restore TGF- ⁇ antiproliferative effect
  • DNA synthesis assay was performed by measuring. the incorporation of [ 3 H]thymidine into the DNA (Fig. 2).
  • [ 3 H] thymidine incorporation into the DNA of Mv1Lu cells was inhibited dose-dependently up to 97% (closed squares), whereas TGF- ⁇ had no effect on the [ 3 H]thymidine incorporation in the R4-2 cells transfected with the vector alone (open squares).
  • T ⁇ R-I/JD1( ⁇ 150-181) The inability of T ⁇ R-I/JD1( ⁇ 150-181) to mediate a growth inhibitory signal raised the possibility that the N-terminal half of the cytoplasmic juxtamembrane domain of T ⁇ R-I contains a site for interaction with downstream component which transduces a signal specific for growth inhibition. Alternatively, such a deletion might change the structural conformation, yielding a receptor which is unable to transduce signals even if the substrate interaction sites were preserved. To address these questions, missense mutations instead of deletion were introduced into certain serine and threonine residues in the T ⁇ R-I juxtamembrane region that was deleted in T ⁇ R-1/JD1( ⁇ 150-181).
  • serine 165, serine 172 and threonine 176 were chosen since these serine and threonine residues were rather conserved among the type I receptors for the TGF- ⁇ superfamily, especially in ActR-IB, which transduces growth inhibition and PAI-1 signals by activin A.
  • Ser and Thr residues were mutated simultaneously or individually to alanine and valine residues, respectively, resulting in four different expression constructs including T ⁇ R-I/JM123(S165A/S172A/T176V), T ⁇ R-I/JM1(S165A), T ⁇ R-I/JM2(S172A) and T ⁇ R-I/JM3 (T176V).
  • TGF- ⁇ binding and physical association with T ⁇ R-II were examined by affinity cross-linking with 125 I-TGF- ⁇ 1 followed by immunoprecipitation using anti-T ⁇ R-II antiserum. All the different receptor mutants were expressed on the cell surface and bound TGF- ⁇ in complex with T ⁇ R-II in a Zn 2+ -dependent manner.
  • T ⁇ R-I/JM1(S165A) Assays for extracellular matrix production and growth inhibition by TGF- ⁇ , the transfected cells were subjected to the analyses for extracellular matrix production and growth inhibition by TGF- ⁇ .
  • PAI-1 and fibronectin assays like wild-type T ⁇ R-I and T ⁇ R-IND1( ⁇ 150-181), all the constructs analyzed including T ⁇ R-I/JM123(S165A/S172A/T176V), T ⁇ R-I/JM1(S165A), T ⁇ RI/JM2(S172A) and T ⁇ R-I/JM3 (T176V) restored responsiveness to TGF- ⁇ .
  • TGF- ⁇ antiproliferative effect the T ⁇ R-I/JM1(S165A) (Fig.
  • T ⁇ R-I and its mutant derivatives in their ability to restore responsiveness to TGF- ⁇ might be due to altered catalytic activity of their receptor kinase.
  • kinase activity was determined by expressing the cytoplasmic regions of T ⁇ R-I and its mutants as GST fusion proteins in E. coli and testing their kinase activities in vitro.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Food Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Led Devices (AREA)
  • Semiconductor Lasers (AREA)
  • Liquid Deposition Of Substances Of Which Semiconductor Devices Are Composed (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Claims (11)

  1. Nucleinsäuremolekül, das für ein Polypeptid kodiert und in einem geeigneten Wirt zu dessen Expression befähigt ist, wobei es sich bei dem Polypeptid um ein Mitglied des Typ I-Rezeptors der TGF-β-Oberfamilie handelt, das die Fähigkeit besitzt, ein zur Matrix-Proteinerzeugung führendes Signal zu transduzieren, das aber eine verringerte Fähigkeit besitzt ein Wachstumshemmsignal zu vermitteln, wobei das Molekül eine Deletion oder Mutation der Wildtyp-Nucleotidsequenz innerhalb eines Bereiches umfasst, der den Aminosäureresten 150-181 von TβR-I entspricht, d. h.
    Figure 00230001
  2. Molekül nach Anspruch 1, wobei die Verringerung auf ein Niveau von nicht mehr als 25 % vorgenommen ist.
  3. Molekül nach Anspruch 1 oder 2, wobei es sich beim Rezeptor um TβR-I handelt.
  4. Molekül nach irgendeinem der vorstehenden Ansprüche, wobei der Bereich den Aminosäureresten 150-176 entspricht.
  5. Molekül nach Anspruch 4, das eine missense-Mutation entsprechend dem Aminosäurerest Serin 172 oder Threonin 176 umfasst.
  6. Polypeptid, umfassend eine Aminosäuresequenz, die durch ein Molekül nach irgendeinem der vorstehenden Ansprüche kodiert wird.
  7. Komplex aus einem Polypeptid nach Anspruch 6 und einem Typ II-Rezeptor der TGF-β-Oberfamilie.
  8. Replizierbarer Expressionsvektor, der in einem transformierten Wirt zur Expression eines Moleküls nach irgendeinem der Ansprüche 1 bis 5 befähigt ist.
  9. Eukaryontische Zelllinie, die mit einem Expressionsvektor nach Anspruch 8 transfiziert ist.
  10. Prokaryontische Zelllinie, die mit einem Expressionsvektor nach Anspruch 8 transformiert ist.
  11. Verwendung eines Moleküls nach irgendeinem der Ansprüche 1 bis 5 zur Identifizierung von Substanzen mit antiproliferativer Aktivität.
EP96929418A 1995-09-04 1996-09-04 Nukleinsäuremolekül, das für einen tgf-rezeptor mit veränderter wachstumshemmung kodiert und seine verwendung Expired - Lifetime EP0848753B1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GBGB9517992.5A GB9517992D0 (en) 1995-09-04 1995-09-04 Growth factors
GB9517992 1995-09-04
PCT/GB1996/002179 WO1997011173A2 (en) 1995-09-04 1996-09-04 Nucleic acid molecule encoding tgf receptor having modified growth inhibition, and its use

Publications (2)

Publication Number Publication Date
EP0848753A2 EP0848753A2 (de) 1998-06-24
EP0848753B1 true EP0848753B1 (de) 2005-01-12

Family

ID=10780148

Family Applications (1)

Application Number Title Priority Date Filing Date
EP96929418A Expired - Lifetime EP0848753B1 (de) 1995-09-04 1996-09-04 Nukleinsäuremolekül, das für einen tgf-rezeptor mit veränderter wachstumshemmung kodiert und seine verwendung

Country Status (7)

Country Link
US (1) US6030795A (de)
EP (1) EP0848753B1 (de)
AT (1) ATE286975T1 (de)
AU (1) AU6883196A (de)
DE (1) DE69634172T2 (de)
GB (1) GB9517992D0 (de)
WO (1) WO1997011173A2 (de)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI815184B (zh) * 2020-09-23 2023-09-11 大陸商海正生物製藥有限公司 Tgfbr2-ecd突變體及包含其的融合蛋白與應用

Also Published As

Publication number Publication date
ATE286975T1 (de) 2005-01-15
US6030795A (en) 2000-02-29
DE69634172D1 (de) 2005-02-17
EP0848753A2 (de) 1998-06-24
WO1997011173A3 (en) 1997-05-09
WO1997011173A2 (en) 1997-03-27
GB9517992D0 (en) 1995-11-08
DE69634172T2 (de) 2006-03-30
AU6883196A (en) 1997-04-09

Similar Documents

Publication Publication Date Title
Escobedo et al. Role of tyrosine kinase and membrane-spanning domains in signal transduction by the platelet-derived growth factor receptor
Braunger et al. Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site
Santoro et al. Constitutive activation of the RON gene promotes invasive growth but not transformation
US5707632A (en) Receptors for fibroblast growth factors
AU702163B2 (en) Morphogenic protein-specific cell surface receptors and uses therefor
Chen et al. Inactivation of the type II receptor reveals two receptor pathways for the diverse TGF-β activities
Wieser et al. Signaling activity of transforming growth factor β type II receptors lacking specific domains in the cytoplasmic region
US6599709B1 (en) Method for screening for soluble ligands to a receptor-type tyrosine kinase
Burke et al. Dimerization of the p185neu transmembrane domain is necessary but not sufficient for transformation
Zhu et al. Receptor chimeras indicate that the met tyrosine kinase mediates the motility and morphogenic responses of hepatocyte growth/scatter factor
JP2002507119A (ja) ヒト受容体型チロシンキナーゼｋｄｒ
US6265160B1 (en) Method of identifying inhibitors of the Jak-Stat signal transduction pathway
EP1403285A1 (de) Rezeptoren der Wachstumsfaktoren aus Fibroblasten
US5789182A (en) System for assaying binding to a heparin-binding growth factor receptor
US6372438B1 (en) Human platelet-derived growth factor receptors
US5863888A (en) Human Bek Fibroblast growth factor receptor
Foehr et al. Discoidin domain receptor 1 (DDR1) signaling in PC12 cells: activation of juxtamembrane domains in PDGFR/DDR/TrkA chimeric receptors
Ihle et al. Signal transduction through the receptor for erythropoietin
Formisano et al. Mutation in a conserved motif next to the insulin receptor key autophosphorylation sites de-regulates kinase activity and impairs insulin action.
EP0848753B1 (de) Nukleinsäuremolekül, das für einen tgf-rezeptor mit veränderter wachstumshemmung kodiert und seine verwendung
JP2002186490A (ja) ヒト血小板由来増殖因子レセプター
US6605703B1 (en) Deletion of the Hck binding region in the IL-6 receptor
Mikami et al. Carboxyl-terminal deletion and point mutations decrease the transforming potential of the activated rat neu oncogene product.
Gardin et al. Substitution of the insulin receptor transmembrane domain with that of glycophorin A inhibits insulin action
Shih et al. Structure and function of ras p21: studies by site-directed mutagenesis

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19980320

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

17Q First examination report despatched

Effective date: 20031009

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20050112

Ref country code: BE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20050112

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20050112

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REF Corresponds to:

Ref document number: 69634172

Country of ref document: DE

Date of ref document: 20050217

Kind code of ref document: P

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20050412

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20050423

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: ISLER & PEDRAZZINI AG

REG Reference to a national code

Ref country code: SE

Ref legal event code: TRGR

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20050930

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20050930

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

ET Fr: translation filed
26N No opposition filed

Effective date: 20051013

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20070830

Year of fee payment: 12

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: IE

Payment date: 20070913

Year of fee payment: 12

REG Reference to a national code

Ref country code: CH

Ref legal event code: PCAR

Free format text: ISLER & PEDRAZZINI AG;POSTFACH 1772;8027 ZUERICH (CH)

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CH

Payment date: 20070830

Year of fee payment: 12

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20050612

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20070829

Year of fee payment: 12

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: SE

Payment date: 20070905

Year of fee payment: 12

Ref country code: NL

Payment date: 20070903

Year of fee payment: 12

Ref country code: IT

Payment date: 20070926

Year of fee payment: 12

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20050112

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20070914

Year of fee payment: 12

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20080904

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20090401

NLV4 Nl: lapsed or anulled due to non-payment of the annual fee

Effective date: 20090401

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

Effective date: 20090529

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20080904

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20080904

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20090401

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20080930

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20080930

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20080930

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20080904

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20080905